The objective of this application is to isolate and study the chemistry and mechanism of action of novel inhibitors of carcinogenesis form higher plants using a bioassay based on B(a)P metabolism. These mammalian cell culture assays detect the inhibitors of interest in a variety of human foods shown to be correlated to reduced cancer incidence. Specific aims include 1) Acquisition of additional higher plants for initial screening in the B(a)P metabolism assay to establish active leads; 2) Bioassay-directed fractionation of plants including members of the Cruciferae, Umbelliferae, Leguminosae, and other s which shown reproducible activity in the bioassays in order to isolate pure, crystalline active inhibitory components. Specific projects underway involve isolation and characterization of active compounds from Trifolium pratense and Coffea arabica. These fractionations will also be guided by assays of the effect of test compounds on benzo(a)pyrene metabolism in hamster embryo cell cultures; 3) Determinations of the complete structure and stereochemistry of crystalline actives using modern spectroscopic (MS), and X-ray crystallography; 4) Determination of the mechanism of action of selected active components with high chemopreventive potential. These studies will include hydrocarbon metabolism, hydrocarbon-DNA interactions and hydrocarbon induced mutation in mammalian systems; and 5) Development of analytical methods including HPLC, GC/MS, and MS/MS to detect these compounds at low levels in biological systems and other plants for purposes of dereplication, identification of other inhibitors, and other plant sources of inhibitors. The identification of these novel inhibitors will lead to a variety of agents with the potential of preventing human cancer, and in addition will provide a better understanding of the total inhibitor activity present in the plants, and their mechanism of action.